The re-emergence of mosquito-borne diseases has challenged the scientific community to develop more effective means for disease control, prevention, and treatment. In order to do this, a more comprehensive understanding of pathogen-host interrelationships during all phases of the pathogen's life cycle must be developed. Lymphatic filariasis is a mosquito-borne disease infecting 120 million people and placing another 1.2 billion at risk. The obligate relationship these nematode parasites have with their mosquito vectors is compromised in certain mosquitoes that mount innate immune responses. Innate immune responses involving the deposition of melanin on the pathogen is the natural resistance mechanism employed by the mosquito Armigeres subalbatus to kill the human filarial worm pathogen, Brugia malayi. But, this mosquito serves as an effective vector for the closely related animal parasite, Brugia pahangi; consequently, the Ar. subalbatus-B. malayi-B. pahangi system, which can be easily manipulated in the laboratory, serves as an ideal model for characterizing innate immune responses that serve as natural determinants of vector competence in mosquito-borne filariasis. Our previous research deliniated the biochemical pathways required for melanin biosynthesis in these defense responses, and new molecular tools and experimental approaches now enable us to begin unravelling the complexities of non-self recognition, target specific melanin deposition, and the genetic regulation/signaling cascades required for this immune response. Herein we propose to use Ar. subalbatus, and the filarial worms B. malayi, B. pahangi and Dirofilaria immitis to profile gene transcription and identify distinct patterns underlying immune responses against filarial worms. These studies will employ oligo-nucleotide microarrays, representing over 6,000 Ar. subalbatus expressed sequence tag (EST) clusters, to temporally compare expression profiles between immune response activated and control mosquitoes and identify key factors involved in this response. Bioinformatics, computational biology, and RNAi will be used to assess genes suspected of influencing the melanization phenotype and to identify key pathways and other factors that might be co-regulated during anti-filarial worm immunity. Proteomics approaches (2-dimensional fluorescence difference gel electrophoresis and isotope-coded affinity tags) with matrix-assisted laser/time-of-flight/mass spectrometry and liquid chromatography/tandem mass spectrometry will be used to temporally profile the hemolymph proteome during responses and to assess post-translational modifications. Mass spectrometry, in concert with affinity isolation techniques, will be used to test the hypothesis that pattern recognition molecules interact with distinct molecules on parasites, mediating further interactions with prophenoloxidase-protein complexes that culminate in the activation of prophenoloxidase and the production of melanin at the site of interaction.
Mosquito-borne lymphatic filariasis extracts a devastating toll on human health, especially amongst less privileged populations in tropical regions of the world. The research described in this proposal will give the scientific community a better understanding of the molecular/genetic factors that control compatible and incompatible relationships between mosquitoes and filarial worm parasites. Understanding these molecular mechanisms could provide clues for new approaches that might be used in the control of mosquito-borne filariasis. ? ? ? ?
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